Plant cells walls are composed mainly of cellulose, pectin, and hemicellulose. Cellulose is comprised of crystalline β-1,4-glucan microfibrils, which are extremely strong and resist enzymatic and mechanical degradation. Cellulose content has a profound effect on the structural properties of plant fibers and wood products, as well as, nutritional quantity, digestibility and palatability of animal and human foodstuffs. Additionally, cellulose is the major structural component of industrially-important plant fibers, such as cotton, flax, hemp, jute and forestry species, such as Eucalyptus ssp. and Pinus ssp.
Cellulose is also commonly used in a variety industrial applications. Some biodegradable plastics and digestible medicine capsules, as well as medical fillers and fiber additives for food can be made from plant polysaccharides. Moreover, certain plastics, such as cellulose acetate, and synthetic textiles, such as rayon, are derived from cellulose.
Polysaccharides have a profound impact on food quality. Cell walls contribute to crispness in carrots, while degradation of cell walls is required for softening of fruits such as peaches and tomatoes. In maize, increased amylose is desirable for cattle feed, but not for human consumption, and increased cell wall strength reduces digestibility. In fiber crops, such as timber, cellulose is the primary polymer of interest. Wood density, a fundamental measure of structural timber quality, is essentially a measure of cellulose content. In the paper pulping industry, efficiency is measured in terms of yield of cellulose and thus a high cellulose content is desirable.
The ability to alter expression of polysaccharide synthesis genes is extremely powerful because polysaccharide synthesis affects plant phenotype as well as growth rates. Control of polysaccharide synthesis has applications for, inter alia, alteration of wood properties and, in particular, lumber and wood pulp properties. For example, improvements to wood pulp that can be effected by altering polysaccharide synthesis gene expression include increased or decreased lignin and cellulose content. Manipulating the polysaccharide synthesis in a plant can also engineer better lumber having increased dimensional stability, increased tensile strength, increased shear strength, increased compression strength, decreased reaction wood, increased stiffness, increased or decreased hardness, decreased spirality, decreased shrinkage, and desirable characteristics with respect to weight, density, and specific gravity.
A. Polysaccharides Genes and Proteins
Cellulose synthesis is catalyzed, in part, by cellulose synthase. Cellulose synthases are members of the large family of inverting processive β-glycosyltransferases. The cellulose synthase (Ces) genes encode cellulose synthases and are responsible, in part, for regulating cellulose biosynthesis. CesA, a cellulose synthase, belongs to the cellulose synthase superfamily, which is characterized by four conserved domains, U1-U4. U1-U3 each have a conserved aspartate as well as an N′ zinc finger domain. The U4 domain possesses a putative substrate binding site, Q-x-x-R—W. Saxena et al., J. Bacteriol. 177: 1419 (1995).
CesA proteins are predicted be an eight transmembrane domain protein having about 1100 amino acids. The CesA proteins function as part of a large membrane-bound complex that polymerizes activated glucose into a cellulose polymer. The substrate for Ces in higher plants is UDP-Glucose (UDPG) and most, if not all evidence supports the hypothesis that cellulose synthase genes encode a glycosyltransferase that is integral to the cellulose biosynthetic pathway (See, Holland et al., Plant Physiol., 123: 1313 (2000)).
In silico analysis identified the cellulose synthase-like proteins (Csl), a large family of proteins in plants believed to be processive polysaccharide β-glycosyltransferases. See, e.g., Goubet et al., Plant Physiol. 131:547 (1993). The cellulose synthase-like proteins possess the conserved U1-U4 domains, like the cellulose synthases, but lack the N′ zinc finger domain. Doblin et al., Plant Cell Physiol. 43:1407 (2002). It is believed that cellulose synthase-like enzymes control the production of non-cellulosic plant polysaccharides.
B. Expression Profiling and Microarray Analysis in Polysaccharide Synthesis
The multigenic control of polysaccharide synthesis presents difficulties in determining the genes responsible for phenotypic determination. One major obstacle to identifying genes and gene expression differences that contribute to phenotype in plants is the difficulty with which the expression of more than a handful of genes can be studied concurrently. Another difficulty in identifying and understanding gene expression and the interrelationship of the genes that contribute to plant phenotype is the high degree of sensitivity to environmental factors that plants demonstrate.
There have been recent advances using genome-wide expression profiling. In particular, the use of DNA microarrays has been useful to examine the expression of a large number of genes in a single experiment. Several studies of plant gene responses to developmental and environmental stimuli have been conducted using expression profiling. For example, microarray analysis was employed to study gene expression during fruit ripening in strawberry, Aharoni et al., Plant Physiol. 129:1019-1031 (2002), wound response in Arabodopsis, Cheong et al., Plant Physiol. 129:661-7 (2002), pathogen response in Arabodopsis, Schenk et al., Proc. Nat'l Acad. Sci. 97:11655-60 (2000), and auxin response in soybean, Thibaud-Nissen et al., Plant Physiol. 132:118. Whetten et al., Plant Mol. Biol. 47:275-91 (2001) discloses expression profiling of cell wall biosynthetic genes in Pinus taeda L. using cDNA probes. Whetten et al. examined genes which were differentially expressed between differentiating juvenile and mature secondary xylem. Additionally, to determine the effect of certain environmental stimuli on gene expression, gene expression in compression wood was compared to normal wood. 156 of the 2300 elements examined showed differential expression. Whetten, supra at 285. Comparison of juvenile wood to mature wood showed 188 elements as differentially expressed. Id. at 286.
Although expression profiling and, in particular, DNA microarrays provide a convenient tool for genome-wide expression analysis, their use has been limited to organisms for which the complete genome sequence or a large cDNA collection is available. See Hertzberg et al., Proc. Nat'l Acad. Sci. 98:14732-7 (2001a), Hertzberg et al., Plant J., 25:585 (2001b). For example, Whetten, supra, states, “A more complete analysis of this interesting question awaits the completion of a larger set of both pine and poplar ESTs.” Whetten et al. at 286. Furthermore, microarrays comprising cDNA or EST probes may not be able to distinguish genes of the same family because of sequence similarities among the genes. That is, cDNAs or ESTs, when used as microarray probes, may bind to more than one gene of the same family.
Methods of manipulating gene expression to yield a plant with a more desirable phenotype would be facilitated by a better understanding of polysaccharide synthetic gene expression in various types of plant tissue, at different stages of plant development, and upon stimulation by different environmental cues. The ability to control plant architecture and agronomically important traits would be improved by a better understanding of how polysaccharide synthesis gene expression effects formation of plant tissues and how plant growth and the polysaccharide synthesis are connected. Among the large number of genes, the expression of which can change during development of a plant, only a fraction are likely to effect phenotypic changes during any given stage of the plant development.